Huanglian ointment alleviates eczema by maintaining the balance of c-Jun and JunB and inhibiting AGE-RAGE-mediated pro-inflammation signaling pathway.

BACKGROUND: Huanglian ointment exhibits clinical efficacy for repairing skin barriers and inhibiting skin inflammation, and has been used to ameliorate eczema for many years. However, the active components and mechanism of Huanglian ointment have not yet been elucidated. PURPOSE: This study aimed to demonstrate the main active components and molecular mechanisms of Huanglian ointment for the treatment of eczema. METHODS: The main active components of Huanglian ointment were identified by gas chromatography-mass spectrometry. Network pharmacology approach and molecular docking techniques to predict the potential molecular mechanisms of Huanglian ointment alleviating eczema. Furthermore, Biostir-AD®-induced guinea pigs and tumor necrosis α (TNF-α)/interferon γ (IFN-γ)-induced HaCaT cells were employed to investigate the effectiveness and mechanisms of Huanglian ointment using histopathological staining, enzyme-linked immunosorbent assay, MTT assay, and western blot analysis. RESULTS: Fourteen chemistry components were identified in Huanglian ointment. In total, 78 intersecting gene targets were identified between Huanglian ointment and eczema, including Jun, inflammatory regulators, and chemokine factors. Intersecting gene targets were enriched for cytokine and chemokine receptor binding, and inflammatory related signaling pathways. The molecular docking results showed that the identified components had a stable binding conformation with core targets. In vivo experiments showed that Huanglian ointment markedly ameliorated eczema-like skin lesions, restored histopathological morphology, and decreased levels of TNF-α, IFN-γ, and interleukin 6. Moreover, Huanglian ointment effectively protected HaCaT cells against TNF-α/IFN-γ-induced cell death and overproduction of thymus- and activation-regulated chemokine, macrophage-derived chemokine, and regulated upon activation normal T cell-expressed and secreted factor. Subsequently, we found that Huanglian ointment repaired skin barriers by affecting c-Jun, JunB, and filaggrin expression, and suppressed inflammatory response by inhibiting AGE-RAGE signaling pathway, both in vivo and in vitro. CONCLUSION: Our results demonstrated that Huanglian ointment repaired skin barriers and inhibited inflammation by maintaining the balance of c-Jun and JunB, and suppressing AGE-RAGE signaling pathway, thereby relieving eczema. These findings providing a molecular basis for treatment of eczema by Huanglian ointment.

Target-guided isolation and purification of antioxidants from Selaginella sinensis by offline coupling of DPPH-HPLC and HSCCC experiments.

Selaginella sinensis (Selaginellaceae) is used extensively in traditional Chinese medicine (TCM) for the treatment of many kinds of chronic diseases. In this study, fractionation of the methanol extract of S. sinensis by different polarity solvents indicated the ethyl acetate fraction exhibited an potent 1,1-diphenyl-2-picryhydrazyl (DPPH) radical scavenging activity with the IC(50) value of 44.9 µM. In order to evaluate the scientific basis, antioxidant peaks were firstly screened using DPPH spiking test through high performance liquid chromatography (DPPH-HPLC). Under the target-guidance of DPPH-HPLC experiment, two flavonoids and six biflavonoids, quercetin (1), apigenin (2), amentoflavone (3), robustaflavone (4), 2,3-dihydroamentaflavone (5), hinokiflavone (6), 4'-O-methyl-robustaflavone (7) and ginkgetin (8) were separated by high-speed counter-current chromatography (HSCCC) method using n-hexane-ethyl acetate-methanol-water (8:8:9:7) as the solvent system with purities 98.2%, 97.6%, 99.4%, 92.3%, 98.5%, 98.9% and 99.6%, respectively. The structures were identified by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Antioxidant activity of eight isolated compounds was assessed by the radical scavenging effect on DPPH radical, compound 1 showed strongest antioxidant activities with IC(50) values of 3.2 µM, while compounds 2-8 showed weak antioxidant activities. This is the first report on simultaneous separation of eight antioxidant compounds from S. sinensis by HSCCC, moreover, apigenin and 4'-O-methyl-robustaflavone were first identified from this plant. Results of the present study indicated that the combinative method using DPPH-HPLC and HSCCC could be widely applied for rapid screening and isolating of antioxidants from complex TCM extract.